Use of aptamers in therapy and/or diagnosis of autoimmune diseases

ABSTRACT

The present invention is directed to an aptamer comprising or consisting of the nucleic acid sequence of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 and/or a nucleic acid sequence being at least 80% identical to one of SEQ ID No. 1, 2 and 3 for use in therapy and/or diagnosis of autoimmune diseases, wherein the autoimmune disease is cardiomyopathy, dilated cardiomyopathy (DCM), peripartum cardiomyopathy (PPCM), idiopathic cardiomyopathy, Chagas&#39; cardiomyopathy, Chagas&#39; megacolon, Chagas&#39; megaesophagus, Chagas&#39; neuropathy, benign prostatic hyperplasia, scleroderma, psoriasis, Raynaud syndrome, pre-eclampsia, kidney allograft rejection, myocarditis, glaucoma, hypertension, pulmonary hypertension, malignant hypertension, and/or Alzheimer&#39;s disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/977,780 filed on Dec. 22, 2015, which is a continuation of U.S.application Ser. No. 14/003,675 filed on Oct. 24, 2013, now U.S. Pat.No. 9,234,201, which is a § 371 national stage entry of InternationalApplication No. PCT/EP2012/053616 filed on Mar. 2, 2012, which claimspriority to European Patent Application No. 11 157 229.3, filed Mar. 7,2011, and U.S. Provisional Patent Application No. 61/449,772, filed Mar.7, 2011, which are hereby incorporated herein by reference in theirentireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 6, 2013, isnamed 3975-85_SL.txt and is 1253 bytes in size.

The immune system forms an essential part of every animal. Mammals makeuse of its immune system in the defence against microorganisms, indetection and removal of aberrant cells like e.g. tumor cells, and inregeneration of tissue. Thereby the organism relies on twointerconnected defence mechanisms, humoral and cellular immunity.

Antibodies, when bound to its antigen are triggers of the humoral immuneresponse. Antibodies can act in multiple ways. Apart from neutralisationof the antigen, antibodies also activate the complement system. Thereare also antibodies which are directed to antigens of the own body. Asreason for the generation of such so called autoantibodies, molecularmimicry and/or bystander activation are seen. Specific binding of theautoantibodies to own antigens can activate natural killer cells (NKcells) which are able to facilitate degradation of these antigens.

Autoimmune diseases are based on such specific recognition and bindingof antibodies directed to own constituent parts of the body whichtriggers an immune response against own cells or tissues. Apart fromthis immunostimmulatory effect, autoantibodies can contribute to thedevelopment of pathogenic phenotypes also by other mechanisms. It iswell known that there are also autoantibodies which can be specific forthe extracellular part of G-protein coupled receptors such as:adrenergic alpha-1 receptor, adrenergic beta-1 receptor, adrenergicbeta-2 receptor, endothelin1 ETA receptor, muscarinic M₂ receptor,angiotensin II AT1 receptor and/or proteinase activated receptors (PAR)receptors and, upon specific binding, can activate or block thesereceptors. The presence of such autoantibodies in an organism can leadto agonistic or antagonistic effects in the sense of a permanentactivation or blockade of the respective receptors which could play arole in the development of the disease.

Dilated cardiomyopathy (DCM) is one of the diseases in that a highpercentage of the patients present with such activating autoantibodiesbinding to extracellular parts of the adrenergic beta-1 receptor, inparticular to the 1st or 2nd loop of adrenergic beta-1 receptor.Consequently, an autoimmune pathogenesis of DCM in these patients wassuggested. Upon binding of these autoantibodies the receptors arecontinuously activated (Jahns et al. (2004) Direct evidence for abeta-1-adrenergic receptor-directed autoimmune attack as a cause ofidiopathic cardiomyopathy. J. Clin. Invest. 113, 1419 to 1429).

In recent studies, it could be shown that removal of theseautoantibodies from the blood via immunoglobulin adsorption contributesto regeneration of the heart muscle (Wallukat G, Reinke P, Dörffel W V,Luther H P, Bestvater K, Felix S B, Baumann G. (1996) Removal ofautoantibodies in dilated cardiomyopathy by immunoadsorption. Int JCardiol. 54:191-195; Müller J, Wallukat G, Dandel M, Bieda H, Brandes K,Spiegelsberger S, Nissen E, Kunze R, Hetzer R (2000) Immunoglobulinadsorption in patients with idiopathic dilated cardiomyopathy.Circulation. 101:385-391. W. V. Dörffel, S. B. Felix, G. Wallukat, S.Brehme, K. Bestvater, T. Hofmann, F K Kleber, G. Baumann, P. Reinke(1997) Short-term hemodynamic effects of immunoadsorption in dilatedcardiomyopathy. Circulation 95, 1994-1997 and W. V. Dörffel, G.Wallukat, Y. Dörffel, S. B. Felix, G. Baumann (2004) Immunoadsorption inidiopathic dilated cardiomyopathy, a 3-year follow-up. Int J. Cardiol.97, 529-534).

There are other diseases of the cardiovascular system which weresuggested to be in relation to the presence of autoantibodies againstG-protein coupled receptors such as, Chagas cardiomyopathy, peripartumcardiomyopathy, myocarditis, pulmonary hypertension and malignanthypertension. Autoantibodies against G-protein coupled receptors werealso found in patients e.g. with glaucoma, Diabetes mellitus, Alzheimerdisease, benign prostatic hyperplasia, scleroderma, Raynaud syndrome,psoriasis, and pre-eclampsia and in chronic Chagas disease as well asthose with kidney allograft rejection.

It is an object of the present invention to provide novel modalities foruse in therapy and/or diagnosis of autoimmune diseases that areassociated with the presence of autoantibodies in the patient.

The present invention provides an aptamer comprising or consisting ofthe nucleic acid sequence of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3and/or a nucleic acid sequence being at least 80% identical to one ofSEQ ID No. 1, 2 and 3 for use in therapy and/or diagnosis of autoimmunediseases.

The aptamers of the invention are characterized in that they comprise orconsist of a nucleic acid sequence of 15 nucleotides with SEQ ID No. 1,SEQ ID No. 2, SEQ ID No. 3 and/or a nucleic acid sequence being at least80% identical to one of SEQ ID No. 1, 2 and 3. The 15-mer: GGT TGG TGTGGT TGG (SEQ ID No. 1), the 26-mer: CGC CTA GGT TGG GTA GGG TGG TGG CG(SEQ ID No. 2) and the 12-mer: GGT TGG TGT GGT (SEQ ID No. 3) are allindependently from each other capable of and responsible for the targetspecificity of the aptamer of the invention. Further nucleic acidmolecules or sequences can be added to the 5′- and/or to the 3′-end ofthe nucleic acid sequence with SEQ ID No. 1, 2 and/or 3. Said 15-mer(SEQ ID No. 1) has first been isolated for its binding to thrombin, seeU.S. Pat. No. 5,543,293, which holds also true for the 26-mer (SEQ IDNo. 2) which was first described in WO/2010/033167. The first mentionedhas already been used under the name ARC183 in clinical phase I trialsfor inhibition of thrombin, i.e. as an anticoagulant for potential usein acute cardiovascular settings. The 26-mer has been used under thename NU172 (ARC 2172) in a clinical phase II trial (clinical trial gov.identifier: NCT 00808964). However, it turned out for the 15-mer (SEQ.ID No. 1) that the amount of aptamer needed to achieve the desiredanticoagulation resulted in a sub-optimal dosing profile.

It has surprisingly been found that the aptamers of the invention can beused to interfere with the interaction of antibodies, in particular ofautoantibodies, specific for G-protein coupled receptors associated withautoimmune diseases. In particular it could be shown that aptamers ofthe invention are capable of binding to autoantibodies specific foradrenergic alpha-1 receptor, adrenergic beta-1 receptor, adrenergicbeta-2 receptor, endothelin 1 ETA receptor, muscarinic M₂ receptor,angiotensin II AT1 receptor, and/or PAR receptors and of inhibiting thespecific interaction of these autoantibodies with its target proteins.By inhibiting these interactions, the aptamers of the invention diminishor even abolish the permanent activation of the respective G-proteincoupled receptors without the need for removal of these antibodies.Thus, the present invention provides compounds that are described thefirst time for their suitability for use in treatment and/or diagnosisof autoimmune diseases, in particular of autoimmune diseases associatedwith the presence of autoantibodies which recognize G-protein coupledreceptors, namely autoimmune diseases associated with the presence ofautoantibodies specific for adrenergic alpha-1 receptor, adrenergicbeta-1 receptor, adrenergic beta-2 receptor, endothelin 1 ETA receptor,muscarinic M₂ receptor, angiotensin II AT1 receptor, and/or PARreceptors. Furthermore after immobilization, the aptamers of theinvention are capable of catching the autoantibiodies indicated above.This way, a platform is provided 1^(st) to establish an apheresistechnology for clearing patient's serum from the autoantibodies and2^(nd) to develop an analytical tool for the measurement of theautoantibodies. The last can be used in particular for diagnosis ofautoimmune diseases.

For the purpose of this invention, the term “aptamer” refers to anoligonucleotide that comprises or consists of the nucleic acid sequenceSEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 and/or a nucleic acid sequencebeing at least 80% identical to one of SEQ ID No. 1, 2 and 3 and iscapable of binding specifically and with high affinity to a particulartarget molecule, e.g. to an autoantibody directed against a G-proteincoupled receptor like e.g. adrenergic alpha-1 receptor, adrenergicbeta-1 receptor, adrenergic beta-2 receptor, endothelin 1 ETA receptor,muscarinic M₂ receptor, angiotensin II AT1 receptor, and/or PARreceptors.

The aptamer of the invention comprises or consists of a sequence ofnucleic acid molecules, the nucleotides. The aptamer of the inventionpreferably comprises unmodified and/or modified D- and/or L-nucleotides.According to the common one letter code of nucleic acid bases “C” standsfor cytosine, “A” stands for adenine, “G” stands for guanine, and “T”stands for thymine, whereas “U” stands for uracil. If not indicatedbelow to the contrary, the term “nucleotide” shall refer toribonucleotides and desoxyribonucleotides. Respectively the terms“2′-fluoro-modified nucleotide”, “2′-methoxy-modified nucleotide”,and/or “2-amino-modified nucleotide” refers to modified ribonucleotidesand modified desoxyribonucleotides.

An aptamer is considered to consist or comprise a nucleic acid sequencebeing at least 80% identical to one of SEQ ID No. 1, 2 and 3, if saidaptamer comprises a contiguous sequence of nucleotides that shows atleast 80% sequence identity over the whole length of SEQ ID No. 1, 2 or3 to the nucleotide sequence of SEQ ID No. 1, 2 or 3, respectively.Means to determine sequence identity are well known in the art and maycomprise e.g. the use of the algorithm blastn.

The aptamer of the invention can comprise a nucleic acid sequence of ≥15nucleotides to ≤160 nucleotides, preferably of ≥15 nucleotides to ≤120nucleotides.

The aptamer of the invention can comprise or consist of a DNA- or anRNA-nucleotide sequence and, thus, can be referred to as DNA-aptamer orRNA-aptamer respectively. It is understood that, if the aptamer of theinvention comprises an RNA-nucleotide sequence, within the sequencemotifs specified throughout the present invention thymin is replaced byuracil. The RNA-nucleotide sequences of the present invention areidentical with the DNA-nucleotide sequences of the invention with theproviso that T is replaced by U. For the sake of conciseness throughoutthe present invention reference is made solely to explicitDNA-nucleotide sequences. However, it is understood that the respectiveRNA-nucleotide sequences are also comprised by the present invention.

The use of DNA-aptamers is particularly preferred. DNA-aptamers areusually more stable in plasma than RNA-aptamers.

The aptamers of the invention may comprise a nucleotide sequencecontaining 2′-modified nucleotides, e.g. 2′-fluoro-, 2′-methoxy- and/or2′-amino-modified nucleotides. The aptamer of the invention may alsocomprise a mixture of desoxyribonucleotides, modifieddesoxyribonucleotides, ribonucleotides and/or modified ribonucleotides.

The aptamer of the invention may comprise modifications. Suchmodifications encompass e.g. alkylation, i.e. methylation, arylation oracetylation of at least one nucleotide, the inclusion of enantiomersand/or the fusion of aptamers with one or more other nucleotides ornucleic acid sequences. Such modifications may comprise e.g. 5′- and/or3′-CAP-modifications or 5′- and/or 3_PEG structures. Alternatively or inaddition the aptamer of the invention may comprise modified nucleotides,preferably selected from locked-nucleic acids, 2′-fluoro-, 2′-methoxy-and/or 2′-amino-modified nucleotides.

Locked nucleic acids (LNA) represent analogons of the respective RNAnucleotides wherein the conformation has been fixed. Oligonucleotides oflocked nucleic acids comprise one or more bicyclic ribonucleosides,wherein the 2′-OH group is connected with the C4-carbon atom via amethylen group. Locked nucleic acids exhibit an improved stabilityversus nucleases compared to the respective unmodified RNA-aptamercounterparts. Also the hybridisation properties are improved whichallows for an enhancement of affinity and specificity of the aptamer.

Another preferred modification is the addition of a so called 3′-CAP-, a5′-CAP-structure and/or of a modified guanosin-nucleotide (e.g.7-methyl-guanosin) to the 3′- and/or 5′-end of the aptamer. Such amodification of the 3′- and/or 5′-end has the effect that the aptamer isprotected from a fast degradation by nucleases.

Alternatively or in addition, the aptamer of the invention can exhibit apegylated 5′-end and/or 3′-end. A 5′-PEG and/or 3′-PEG modificationcomprises the addition of at least one polyethylene glycol (PEG) unit,preferably the PEG group comprises 1 to 900 ethylene groups, morepreferably from 1 to 450 ethylene groups. In a preferred embodiment, theaptamer comprises linear PEG units with HO—(CH₂CH₂O)_(n)—H, wherein n isan integer of 1 to 900, preferably n is an integer of 1 to 450.

The aptamer of the invention can comprise or consist of a nucleic acidsequence with a phospho-thioate backbone or can be wholly or in partconfigured as a peptide nucleic acid (PNA).

One advantage of modifying the aptamer of the invention by one or moreof the ways mentioned above is that the aptamer can be stabilizedagainst detrimental influences like e.g. nucleases present in theenvironment wherein the aptamer is used. Said modifications are alsosuitable to adapt the pharmacological properties of the aptamer. Themodifications preferably do not alter the affinity or specificity of theaptamer.

The aptamer of the invention may also be conjugated to a carriermolecule and/or to a reporter molecule. Carrier molecules comprise suchmolecules that, when conjugated to the aptamer, prolong the plasma halflife of the conjugated aptamer in human plasma, e.g. by enhancing thestability and/or by affecting the excretion rate. One example of asuitable carrier molecule is PEG. Reporter molecules comprise moleculesthat allow for the detection of the conjugated aptamer. Examples of suchreporter molecules are GFP, biotin, cholesterol, dyes like e.g.fluorescence dyes, electrochemically active reporter molecules and/orcompounds comprising radioactive residues, in particular radionuclidessuitable for PET (positron emission tomography) detection like e.g. ¹⁸F,¹¹C, ¹³N, ¹⁵O, ⁸²Rb or ⁶⁸Ga. The skilled person is well aware ofsuitable carrier and reporter molecules and of ways of how to conjugatethem to the aptamer of the invention.

The aptamer of the invention inhibits the agonistic or blocking effectof an antibody. For the purpose of the present invention, the term“antibody” refers to naturally occurring antibodies, including e.g.autoantibodies in particular autoantibodies of a patient suffering froman autoimmune disease related to the presence of autoantibodies specificfor a G-protein coupled receptor like e.g. adrenergic alpha-1 receptor,adrenergic beta-1 receptor, adrenergic beta-2 receptor, endothelin 1 ETAreceptor, muscarinic M₂ receptor, angiotensin II AT1 receptor, and/orPAR receptors, and modified or genetically engineered antibodies. Anautoantibody is an antibody manufactured by the immune system of anindividual that is directed against one or more of the individual's ownproteins. However, the term antibody is not limited to an antibody withthe classical heavy and light chain architecture. The terms “antibody”or “antibodies” as used herein are art-recognized terms and areunderstood to refer to molecules or active fragments of molecules thatbind to given antigens, particularly the terms refer to immunoglobulinmolecules and to immunologically active portions of immunoglobulinmolecules, i.e. molecules that contain a binding site that specificallybinds an antigen. An immunoglobulin is a protein comprising one or morepolypeptides substantially encoded by the immunoglobulin kappa andlambda, alpha, gamma, delta, epsilon and mu constant region genes, aswell as myriad immunoglobulin variable region genes. Light chains areclassified as either kappa or lambda. Heavy chains are classified asgamma, mu, alpha, delta, or epsilon, which in turn define theimmunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. Alsosubclasses of the heavy chain are known. For example, IgG heavy chainsin humans can be any of IgG1, IgG2, IgG3 and IgG4 subclass. The antibodycan preferably be of IgM and/or IgG class or any subclass thereof (IgG1,IgG2, IgG3, IgG4).

“Antibodies” are intended within the scope of the present invention toinclude autoantibodies, monoclonal antibodies, polyclonal antibodies,chimeric, single chain, bispecific, simianized, human and humanizedantibodies as well as active fragments thereof. Examples of activefragments of molecules that bind to known antigens include separatedlight and heavy chains, Fab, Fab/c, Fv, Fab′, and F(ab′)2 fragments,including the products of an Fab immunoglobulin expression library andepitope-binding fragments of any of the antibodies and fragmentsmentioned above.

The aptamers of the invention are used in treatment and/or diagnosis ofautoimmune diseases. As used for the purpose of the present invention,the term “autoimmune disease” or “autoimmune diseases” refers toautoimmune diseases, in particular to autoimmune diseases in a human,wherein the autoimmune diseases are associated with the presence ofautoantibodies specific for a G-protein coupled receptor. Saidautoantibodies may preferably be involved in the pathogenesis of theautoimmune disease and, as such, may be present in the serum of apatient suffering from said autoimmune disease. More preferably theautoimmune diseases are autoimmune diseases associated with the presencein the serum of the patient of autoantibodies specific for adrenergicalpha-1 receptor, adrenergic beta-1 receptor, adrenergic beta-2receptor, endothelin 1 ETA receptor, muscarinic M₂ receptor, angiotensinII AT1 receptor, and/or PAR receptors. Even more preferred, theautoimmune disease is cardiomyopathy, dilated cardiomyopathy (DCM),peripartum cardiomyopathy (PPCM), idiopathic cardiomyopathy, Chagas'cardiomyopathy, Chagas' megacolon, Chagas' megaesophagus, Chagas'neuropathy, benign prostatic hyperplasia, scleroderma, psoriasis,Raynaud syndrome, pre-eclampsia, kidney allograft rejection,myocarditis, glaucoma, hypertension, pulmonary hypertension, malignanthypertension, and/or Alzheimer's disease. Most preferably the term“autoimmune disease” or “autoimmune diseases” refers to the autoimmunediseases dilated cardiomyopathy (DCM), peripartum cardiomyopathy (PPCM),Chagas' cardiomyopathy, Chagas' megacolon, glaucoma, hypertension,pulmonary hypertension, malignant hypertension, kidney allograftrejection, Raynaud syndrome and/or Alzheimer's disease.

The manufacturing or mass production of aptamers of the invention iswell known in the art and represents a mere routine activity.

The present invention is also directed to a pharmaceutical compositioncomprising at least one aptamer of the invention and, optionally, atleast one pharmaceutically acceptable excipient. The invention is alsodirected to a pharmaceutical composition comprising an aptamer of theinvention or a mixture of different aptamers of the invention and apharmaceutically acceptable excipient like e.g. a suitable carrier ordiluent.

Preferably the aptamer of the invention constitutes an active ingredientof the pharmaceutical composition and/or is present in an effectiveamount.

The term “effective amount” denotes an amount of the aptamer of theinvention having a prophylactically, diagnostically or therapeuticallyrelevant effect on a disease or pathological conditions. A prophylacticeffect prevents the outbreak of a disease. A therapeutically relevanteffect relieves to some extent one or more symptoms of a disease orreturns to normal either partially or completely one or morephysiological or biochemical parameters associated with or causative ofthe disease or pathological conditions. The respective amount foradministering the aptamer of the invention is sufficiently high in orderto achieve the desired prophylactic, diagnostic or therapeutic effect.It will be understood by the skilled person that the specific doselevel, frequency and period of administration to any particular mammalwill depend upon a variety of factors including the activity of thespecific components employed, the age, body weight, general health, sex,diet, time of administration, route of administration, drug combination,and the severity of the specific therapy. Using well-known means andmethods, the exact amount can be determined by one of skill in the artas a matter of routine experimentation.

In the pharmaceutical composition of the invention at least 20% of thetotal aptamer content is made of an aptamer of the invention, preferablyat least 50%, more preferably at least 75%, most preferable at least95%.

When used for therapy, the pharmaceutical composition will generally beadministered as a formulation in association with one or morepharmaceutically acceptable excipients. The term “excipient” is usedherein to describe any ingredient other than the aptamer of theinvention. The choice of excipient will to a large extent depend on theparticular mode of administration. Excipients can be suitable carriersand/or diluents.

The pharmaceutical composition of the invention may be administeredorally. Oral administration may involve swallowing, so that thecomposition enters the gastrointestinal tract, or buccal or sublingualadministration may be employed by which the composition enters the bloodstream directly from the mouth.

Formulations suitable for oral administration include: solidformulations such as tablets; coated tablets, capsules containingparticulates, liquids, or powders; lozenges (including liquid-filled);and chews; multi- and nano-particulates; gels; solid solutions;liposomes; films, ovules, sprays and liquid formulations.

Liquid formulations include suspensions, solutions, syrups and elixirs.Such formulations may be employed as fillers in soft or hard capsulesand typically comprise a carrier, for example, water, ethanol,polyethylene glycol, propylene glycol, methylcellulose, or a suitableoil, and one or more emulsifying agents and/or suspending agents. Liquidformulations may also be prepared by the reconstitution of a solid, forexample, from a sachet.

For tablet dosage forms, depending on dose, the aptamer of the inventionmay make up from 0.1 weight % to 80 weight % of the dosage form, moretypically from 5 weight % to 60 weight % of the dosage form. In additionto the aptamer of the invention, tablets generally contain adisintegrant. Examples of disintegrants include sodium starch glycolate,sodium carboxymethyl cellulose, calcium carboxymethyl cellulose,croscarmellose sodium, crospovidone, polyvinylpyrrolidone, methylcellulose, microcrystalline cellulose, lower alkyl-substitutedhydroxypropyl cellulose, starch, pregelatinised starch and sodiumalginate. Generally, the disintegrant will comprise from 1 weight % to25 weight %, preferably from 5 weight % to 20 weight % of the dosageform.

Tablets may comprise additional excipients like e.g. binders, surfaceactive agents, lubricants and/or other possible ingredients like e.g.anti-oxidants, colorants, flavouring agents, preservatives and/ortaste-masking agents.

Tablet blends may be compressed directly or by roller to form tablets.Tablet blends or portions of blends may alternatively be wet-, dry-, ormelt-granulated, melt congealed, or extruded before tabletting. Thefinal formulation may comprise one or more layers and may be coated oruncoated; it may even be encapsulated.

Solid formulations for oral administration may be formulated to beimmediate and/or modified release. Modified release formulations includedelayed-, sustained-, pulsed-, controlled-, targeted and programmedrelease.

The pharmaceutical composition of the invention may also be administereddirectly into the blood stream, into muscle, or into an internal organ.Suitable means for parenteral administration include intravenous,intraarterial, intraperitoneal, intrathecal, intraventricular,intraurethral, intrasternal, intracranial, intramuscular andsubcutaneous. Suitable devices for parenteral administration includeneedle (including microneedle) injectors, needle-free injectors andinfusion techniques.

Parenteral formulations are typically aqueous solutions which maycontain excipients such as salts, carbohydrates and buffering agents(preferably to a pH of from 3 to 9), but, for some applications, theymay be more suitably formulated as a sterile non-aqueous solution or asa dried form to be used in conjunction with a suitable vehicle such assterile, pyrogen-free water.

The preparation of parenteral formulations under sterile conditions, forexample, by lyophilisation, may readily be accomplished using standardpharmaceutical techniques well known to those skilled in the art.

The solubility of pharmaceutical composition of the invention used inthe preparation of parenteral solutions may be increased by the use ofappropriate formulation techniques, such as the incorporation ofsolubility-enhancing agents.

Formulations for parenteral administration may be formulated to beimmediate and/or modified release. Modified release formulations includedelayed-, sustained-, pulsed-, controlled-, targeted and programmedrelease. Thus compounds of the invention may be formulated as a solid,semi-solid, or thixotropic liquid for administration as an implanteddepot providing modified release of the active compound. Examples ofsuch formulations include drug-coated stents andPGLApoly(dl-lactic-coglycolic)acid (PGLA) micro spheres.

The pharmaceutical composition of the invention may also be administeredtopically to the skin or mucosa, that is, dermally or transdermallyTypical formulations for this purpose include gels, hydrogels, lotions,solutions, creams, ointments, dusting powders, dressings, foams, films,skin patches, wafers, implants, sponges, fibres, bandages andmicroemulsions. Liposomes may also be used. Typical carriers includealcohol, water, mineral oil, liquid petrolatum, white petrolatum,glycerin, polyethylene glycol and propylene glycol. Penetrationenhancers may be incorporated. Other means of topical administrationinclude delivery by electroporation, iontophoresis, phonophoresis,sonophoresis and microneedle or needle-free (e.g. Powderject™, Bioject™,etc.) injection. Formulations for topical administration may beformulated to be immediate and/or modified release. Modified releaseformulations include delayed-, sustained-, pulsed-, controlled-,targeted and programmed release.

For administration to human patients, the total daily dose of theaptamer of the invention and/or the pharmaceutical composition of theinvention is typically in the range 0.001 mg to 5000 mg depending, ofcourse, on the mode of administration. For example, an intravenous dailydose may only require from 0.001 mg to 40 mg. The total daily dose maybe administered in single or divided doses and may, at the physician'sdiscretion, fall outside of the typical range given herein.

These dosages are based on an average human subject having a weight ofabout 65 kg to 70 kg. The physician will readily be able to determinedoses for subjects whose weight falls outside this range, such asinfants and the elderly.

The present invention also encompasses a kit comprising an aptamer ofthe invention, a pharmaceutical composition, a container and optionallywritten instructions for use and/or with means for administration.

The aptamer, the pharmaceutical composition and/or the kit of theinvention are used in therapy and/or diagnosis of autoimmune diseases,in particular of autoimmune diseases in a human. Preferably, theautoimmune diseases are autoimmune diseases associated with the presenceof autoantibodies specific for a G-protein coupled receptor, wherein theautoantibodies are present in the serum of a patient suffering from saidautoimmune disease. More preferably the autoimmune diseases areautoimmune diseases associated with the presence in the serum of thepatient of autoantibodies specific for adrenergic alpha-1 receptor,adrenergic beta-1 receptor, adrenergic beta-2 receptor, endothelin 1 ETAreceptor, muscarinic M₂ receptor, angiotensin II AT1 receptor, and/orPAR receptors. Even more preferred, the autoimmune disease iscardiomyopathy, dilated cardiomyopathy (DCM), peripartum cardiomyopathy(PPCM), idiopathic cardiomyopathy, Chagas' cardiomyopathy, Chagas'megacolon, Chagas' megaesophagus, Chagas' neuropathy, benign prostatichyperplasia, scleroderma, psoriasis, Raynaud syndrome, pre-eclampsia,kidney allograft rejection, myocarditis, glaucoma, hypertension,pulmonary hypertension, malignant hypertension, and/or Alzheimer'sdisease. Most preferably the term “autoimmune disease” or “autoimmunediseases” refers to the autoimmune diseases dilated cardiomyopathy(DCM), peripartum cardiomyopathy (PPCM), Chagas' cardiomyopathy, Chagas'megacolon, glaucoma, hypertension, pulmonary hypertension, malignanthypertension, kidney allograft rejection, Raynaud syndrome and/orAlzheimer's disease.

The terms “therapy”, “treatment” and “therapeutically,” as used herein,refer to the act of treating, as “treating” is defined below. As usedherein, the term “treating” refers to reversing, alleviating orinhibiting the progress of a disease, disorder or condition, or one ormore symptoms of such disease, disorder or condition, to which such termapplies. As used herein, “treating” may also refer to decreasing theprobability or incidence of the occurrence of a disease, disorder orcondition in a mammal as compared to an untreated control population, oras compared to the same mammal prior to treatment. For example, as usedherein, “treating” may refer to preventing a disease, disorder orcondition, and may include delaying or preventing the onset of adisease, disorder or condition, or delaying or preventing the symptomsassociated with a disease, disorder or condition. As used herein,“treating” may also refer to reducing the severity of a disease,disorder or condition or symptoms associated with such disease, disorderor condition prior to a mammal's affliction with the disease, disorderor condition. Such prevention or reduction of the severity of a disease,disorder or condition prior to affliction relates to the administrationof the composition of the present invention, as described herein, to asubject that is not at the time of administration afflicted with thedisease, disorder or condition. As used herein “treating” may also referto preventing the recurrence of a disease, disorder or condition or ofone or more symptoms associated with such disease, disorder orcondition.

For treatment and/or diagnosis of a disease, irrespective of the routeof administration, the aptamer of the invention is administered at adaily dose per treatment cycle of not more than 20 mg/kg body weight,preferably of not more than 10 mg/kg body weight, more preferablyselected from the range of 1 μg/kg to 20 mg/kg body weight, mostpreferably selected from a range of 0.01 to 10 mg/kg body weight.

The present invention is also directed to the use of an aptamer of theinvention or pharmaceutical composition of the invention in themanufacture of a medicament for treatment and/or diagnosis of anautoimmune disease. Preferably the autoimmune disease is cardiomyopathy,dilated cardiomyopathy (DCM), peripartum cardiomyopathy (PPCM),idiopathic cardiomyopathy, Chagas' cardiomyopathy, Chagas' megacolon,Chagas' megaesophagus, Chagas' neuropathy, benign prostatic hyperplasia,scleroderma, psoriasis, Raynaud syndrome, pre-eclampsia, kidneyallograft rejection, myocarditis, glaucoma, hypertension, pulmonaryhypertension, malignant hypertension, and/or Alzheimer's disease. Evenmore preferably the term “autoimmune disease” or “autoimmune diseases”refers to the autoimmune diseases dilated cardiomyopathy (DCM),peripartum cardiomyopathy (PPCM), Chagas' cardiomyopathy, Chagas'megacolon, glaucoma, hypertension, pulmonary hypertension, malignanthypertension, kidney allograft rejection, Raynaud syndrome and/orAlzheimer's disease.

In another aspect, the present invention is directed to a method oftreatment or diagnosis of an autoimmune disease, wherein an individualin need of such treatment is administered an effective amount of anaptamer of the invention or a pharmaceutical composition of theinvention. Preferably the autoimmune disease is cardiomyopathy, dilatedcardiomyopathy (DCM), peripartum cardiomyopathy (PPCM), idiopathiccardiomyopathy, Chagas' cardiomyopathy, Chagas' megacolon, Chagas'megaesophagus, Chagas' neuropathy, benign prostatic hyperplasia,scleroderma, psoriasis, Raynaud syndrome, pre-eclampsia, kidneyallograft rejection, myocarditis, glaucoma, hypertension, pulmonaryhypertension, malignant hypertension, and/or Alzheimer's disease. Evenmore preferably the term “autoimmune disease” or “autoimmune diseases”refers to the autoimmune diseases dilated cardiomyopathy (DCM),peripartum cardiomyopathy (PPCM), Chagas' cardiomyopathy, Chagas'megacolon, glaucoma, hypertension, pulmonary hypertension, malignanthypertension, kidney allograft rejection, Raynaud syndrome and/orAlzheimer's disease.

The aptamer of the invention, when used for treatment or diagnosis of anautoimmune disease does not necessarily need to be administered to anindividual or patient. The therapeutic or diagnostic effect may also beachieved by use of the aptamer of the invention for elimination ofantibodies, like e.g. autoantibodies from the body or from body fluids.Such an elimination may comprise the application of the aptamer of theinvention in a setting where the aptamer of the invention is contactedwith a body fluid solely ex vivo, e.g. during immune adsorption and/orapheresis, so that the aptamer of the invention does not enter the bodyof the individual or patient to be treated. Thus, the present inventionis also directed to an apheresis column comprising an aptamer of theinvention.

Apheresis is a medical technology in which the blood of a donor orpatient is passed through an apparatus that separates out one particularconstituent and returns the remainder back to the circulation of thedonor or patient. The aptamer of the invention can be used as selectiveingredient during apheresis. The selective ingredient is responsible forspecifically separating out the desired particular constituents, namelythe antibodies or autoantibodies present in the sample or blood whichare specifically targeted by the aptamer of the invention. Preferablythe aptamer of the invention is used as selective ingredient intherapeutic apheresis of blood or parts thereof derived from a patientsuffering from an autoimmune disease. More preferably the autoimmunedisease is cardiomyopathy, dilated cardiomyopathy (DCM), peripartumcardiomyopathy (PPCM), idiopathic cardiomyopathy, Chagas'cardiomyopathy, Chagas' megacolon, Chagas' megaesophagus, Chagas'neuropathy, benign prostatic hyperplasia, scleroderma, psoriasis,Raynaud syndrome, pre-eclampsia, kidney allograft rejection,myocarditis, glaucoma, hypertension, pulmonary hypertension, malignanthypertension, and/or Alzheimer's disease. Even more preferably the term“autoimmune disease” or “autoimmune diseases” refers to the autoimmunediseases dilated cardiomyopathy (DCM), peripartum cardiomyopathy (PPCM),Chagas' cardiomyopathy, Chagas' megacolon, glaucoma, hypertension,pulmonary hypertension, malignant hypertension, kidney allograftrejection, Raynaud syndrome and/or Alzheimer's disease.

The present invention also relates to an aptamer of the inventioncoupled to a solid support. The skilled person is well aware oftechniques and materials which may be used to produce such aptamerscoupled to a solid support. In a preferred embodiment, the solid supportcomprises a solid material that is applicable in medical, biochemical orbiological assays, like e.g. materials used in apheresis or ELISAassays. Said solid material comprises polymers that are usually used assupport in medical, biochemical or biological assays. In particular theaptamer of the invention may be coupled to a solid support that allowsfor the use of the resulting product in the manufacturing of a columnsuitable for apheresis, preferably a column that is suitable for use inan apheresis to remove antibodies that can be specifically bound by theaptamer of the invention from a liquid sample, preferably from a bodyfluid.

In a further aspect, the present invention is directed to the use of anaptamer of the invention for the in vitro detection and/orcharacterization of antibodies, like e.g. autoantibodies, being specificfor a G-protein coupled receptor, preferably the G-protein coupledreceptors adrenergic alpha-1 receptor, adrenergic beta-1 receptor,adrenergic beta-2 receptor, endothelin 1 ETA receptor, muscarinic M₂receptor, angiotensin II AT1 receptor, and/or PAR receptors.

Such a use may comprise the testing of a sample in a rat cardiomyocytebeating frequency assay in the presence and absence of an effectiveamount of an aptamer of the invention. Depending on the effect of thesample and the aptamer of the invention on the beating frequency, theskilled person can conclude on the presence of respective antibodies.Data on total or relative quantity of such antibodies in the sample mayalso be gained as well as on other properties of such antibodies.

The so called rat cardiomyocyte beating frequency assay is a wellestablished assay for detection and characterization of antibodies, e.g.autoantibodies derived from patients, specific for a number of humanG-protein coupled receptors like e.g. human adrenergic alpha-1 receptor,adrenergic beta-1 receptor, adrenergic beta-2 receptor, endothelin 1 ETAreceptor, muscarinic M₂ receptor, angiotensin II AT1 receptor, and/orPAR receptors. The assay is described in detail in Wallukat et al.(1987) Effects of the serum gamma globulin fraction of patients withallergic asthma and dilated cardiomyopathy on chromotropic betaadrenoceptor function in cultured neonatal rat heart myocytes, Biomed.Biochim. Acta 46, 634-639; Wallukat et al. (1988) Cultivated cardiacmuscle cells—a functional test system for the detection ofautoantibodies against the beta adrenergic receptor, Acta Histochem.Suppl. 35, 145-149; and Wallukat et al. (2010) Distinct patterns ofautoantibodies against G-protein coupled receptors in Chagas'cardiomyopathy and megacolon. Their potential impact for early riskassessment in asymptomatic Chagas' patients, J. Am. Coll. Cardiol. 55,463-468. Thus, the skilled person is well aware of the nature of thisassay and knows how to apply it.

The aptamer of the invention can particularly be used in detectionand/or characterization of respective antibodies, wherein the antibodiesare presented in or derived from a body fluid, preferably a fluid of thehuman body, more preferably of human blood, plasma, serum, urine, feces,synovial fluid, interstitial fluid, lymph, saliva, sudor, spinal fluidand/or lacrimal fluid. In a preferred embodiment, the body fluid istaken from an individual suffering from or suspected to suffer from anautoimmune disease. Preferably the autoimmune disease is cardiomyopathy,dilated cardiomyopathy (DCM), peripartum cardiomyopathy (PPCM),idiopathic cardiomyopathy, Chagas' cardiomyopathy, Chagas' megacolon,Chagas' megaesophagus, Chagas' neuropathy, benign prostatic hyperplasia,scleroderma, psoriasis, Raynaud syndrome, pre-eclampsia, kidneyallograft rejection, myocarditis, glaucoma, hypertension, pulmonaryhypertension, malignant hypertension, and/or Alzheimer's disease. Evenmore preferably the term “autoimmune disease” or “autoimmune diseases”refers to the autoimmune diseases dilated cardiomyopathy (DCM),peripartum cardiomyopathy (PPCM), Chagas' cardiomyopathy, Chagas'megacolon, glaucoma, hypertension, pulmonary hypertension, malignanthypertension, kidney allograft rejection, Raynaud syndrome and/orAlzheimer's disease.

For detection and/or characterization of such antibodies, the aptamer ofthe invention may be used in solution or in an immobilized form.

The aptamer of the invention may be used for direct or indirectdetection and/or characterization of said antibodies.

In the following the invention will be further explained and exemplifiedby way of examples.

FIGURES

FIG. 1 shows the dose-response curves of the neutralization of thefunctional activity of different AABs as indicated in the figure.

FIG. 2 shows the influence of the presence of human IgG-3 [73 nM] on thedose-response curve neutralizing the functional activity ofbeta1-receptor AABs by the thrombin-aptamer.

FIG. 3 shows the influence of the thrombin-aptamer on the ETA-ABmediated decrease of the beating frequency of neonatale cardiomyocytes.

FIGS. 4A, 4B, 4C and 4D show the binding of the thrombin-aptamer (SEQ.ID No. 1) on the immobilized ETA-AB, the immobilized rabbit IgG and theimmobilized human IgG-subclasses for control. 4A and 4C: 25 nM of eachprotein immobilized, 4B and 4D: 250 nM of the proteins immobilized. 4Aand 4B: binding of the thrombin-aptamer SEQ. ID No. 1 and 4C and 4D:binding of its scrambled control sequence. The biotinylatedthrombin-aptamer was used and the biotin moiety served for detection.The amount of bound biotin was quantified via Neutravidin-POD and theTMB/H₂O₂-reaction.

FIGS. 5A and 5B show the binding of the ETA-AB (SP 4122P) ontoimmobilized thrombin-aptamer (SEQ. ID No. 1) 5A: 1 μM 5B: 0.1 μMthrombin-aptamer for immobilization. For detection served the secondaryanti-rabbit IgG-POD antibody (1:10.000) and the TMB/H₂O₂-reaction. Theuncoated and the Neutravidine-coated plate served for control.(Neutravidine=NA).

FIG. 6 shows the binding of the ETA-AB (SP 4122P) onto immobilizedthrombin-aptamer (SEQ. ID No. 1) and the immobilized scrambledthrombin-aptamer for control.

FIG. 7 shows the recovery of the bound ETA-AB after elution from thethrombin-aptamer column and the corresponding control experiment(control column). The ETA-AB activity was measured in the bioassay.

FIGS. 8A and 8B show the recovery of the ETA-AB from the spiked serum inthe ELISA experiment. 8A: shows the ETA-AB standard curve which wastreated comparably to the eluate samples (dialysis). 8B: shoes theamount of recovered ETA-AB in the flow-throughs, the washing solutionand the eluates after elution from the thrombin-aptamer column (ARC183column) and the corresponding control column (scrambledthrombin-aptamer). For detection served an anti-rabbit-IgG-POD antibody(1:10,000) and the TMB/H₂O₂ detection.

FIG. 9 shows the testing of AAB-neutralization capacity of the5′-FITC-labelled thrombin-aptamer using the bioassay of spontaneouslybeating neonatale rat cardiomyocytes. The increase of beating frequencycaused by beta1-receptor AABs was reduced by about 50% when 100 nMFITC-thrombin-aptamer was present. The bars are the mean of twoindependent experiments (n=2).

FIG. 10 shows the detection of ETA-AAB of a patient sample using anthrombin-aptamer//FITC-thrombin-aptamer sandwich assay. For controlserved a control IgG sample and scramble thrombin-aptamer. The data arefrom one experiment. (FITC-throm-a=FITC-thrombin-aptamer,throm-apta=thrombin-aptamer).

FIG. 11 shows the neutralization of AAB activity (beta1-receptor AAB,alpha1+beta2-receptor AAB, affinity purified beta2-receptor AAB, eachAAB n=1) by 100 nM dT-thrombin-aptamer, measured in the bioassay ofspontaneously beating neonatale cardiomyocytes.

FIG. 12 shows testing of the functionality of the truncatedthrombin-aptamer sequence (12-mer sequence, SEQ ID No. 3, Throm K1)compared to the original 15-mer sequence (ARC183, SEQ ID No. 1) and the26-mer sequence (NU172, SEQ ID No. 2) neutralizing the positivechronotropic activity of beta1-receptor AABs in the bioassay ofspontaneously beating rat cardiomyocytes.

EXAMPLES Summary

The aptamers consisting of a nucleic acid sequence with SEQ. ID No. 1and 2, respectively, are affine and specific binders for autoantibodieswhich target the G-protein coupled receptors. The aptamers are able toneutralize the AAB (autoantibody)-function in the soluble state.Immobilized onto surfaces the aptamers were able to capture AABs. Afollowing elution will remove the captured AABs.

It has therefore been shown that aptamers of the present invention areappropriate molecules for therapeutic and diagnostic purposes for thetreatment of diseases which are associated to functional active AABsagainst G-protein coupled receptors.

Material and Methods

Cardiomyocyte Preparation and Culturing

Hearts of 1 to 3 day old rats were removed under sterile conditions andtransferred to phosphate buffered saline solution (PBS) containingpenicillin/streptomycin. The ventricle tissue was separated, dissectedin pieces and washed twice with 10 ml PBS containingpenicillin/streptomycin and at last once with PBS only. The ventriclepieces were suspended in 30 ml PBS containing 0.2% trypsin. Afterincubation for 20 min at 37° C., the trypsination was stopped with 10 mlice-cold heat-inactivated calf serum. The resulting suspension wascentrifuged at 130×g for 6 min and the pellet transferred to 20 ml ofSM20-I medium. For cell count estimation, 100 μl of this suspension wasadded to 100 μl trypanblue solution. For cell culturing 2.4×10⁶ cellswere suspended in 2.5 ml of glucose containing SM 20-I medium which wasequilibrated with humid air and supplemented with 10% heat-inactivatedcalf serum, 0.1 mU insulin, and 2 μM fluorodeoxyuridine (preventing theovergrowth of the myocytes by non-myocytes), transferred to 12.5-cm²Falcon flasks and cultured as monolayers for 4 days at 37° C. The mediumwas renewed after two days.

Bioassay Principle

For the identification and quantification of the AAB, a bioassay wasused which was firstly established for the measurement of AAB againstG-protein coupled receptors by Wallukat and Wollenberger (Wallukat G,Wollenberger A. Effects of the serum gamma globulin fraction of patientswith allergic asthma and dilated cardiomyopathy on chromotropic betaadrenoceptor function in cultured neonatal rat heart myocytes. BiomedBiochim Acta 1987; 46:S634-9) and which we recently described in detailfor the measurement of AAB against the adrenergic beta1 and beta2- andthe muscarinergic M₂-receptors in chronic Chagas disease (Wallukat G,Muñoz Saravia S G, Haberland A, Bartel S, Araujo R, Valda G, Duchen D,Diaz Ramirez I, Borges A C, Schimke I. Distinct patterns ofautoantibodies against G-protein-coupled receptors in Chagas'cardiomyopathy and megacolon. Their potential impact for early riskassessment in asymptomatic Chagas' patients. J Am Coll Cardiol. 2010;55:463-8.).

In this bioassay, the chronotropic response of spontaneously beatingcultured neonatal rat cardiomyocytes was recorded which is the sum ofpositive chronotropy caused by stimulating AAB such as the onestargeting adrenergic beta1- and beta2-receptors or the adrenergicalpha1-receptor and negative chronotropy caused by inhibiting AAB suchas the ones targeting muscarinergic M₂-receptors or the endothelinreceptor type A (ETA-receptor) (1 unit of AAB activity=1 beat/minfrequency change).

To differentiate the AAB species with respect to their contribution tothe chronotropic response (positive or negative chronotropy), theanalysis was conducted in the presence of specific antagonists such asICI-118.551 for beta2-receptor AAB, atropine for M₂-AAB, propranolol forbeta1/beta2-receptor AAB, BQ 610 or BQ 123 for the ETA-receptor,prazosine for the alpha1-adrenoceptor and Ibesartan or Losartan for theAT1-receptor. The remaining activity change is caused by AAB except theonce which were specifically blocked.

A further characterization of different receptor AABs demonstrated abovewas done by using the AAB-epitope-representing-peptides corresponding tothe extracellular loops of the receptors.

The bioassay can detect and quantify all human serum AAB and othermolecules which target receptors on the cell surface whose sequences arehomologue to the human receptors (in the case of AAB targeting) andwhich are linked to a machinery regulating the beating frequency(contractility, chronotropy) of the cells such as the G-protein system.

Preparation of Serum Samples for AAB Identification and Differentiationand AAB Activity Measurement

1 ml of control and patient serum and 660 μl saturated ammonium sulfatesolution were mixed (final concentration 40% ammonium sulphate) andincubated for 18 hours at 4° C. After centrifugation for 15 min at6,000×g, the pellet was re-suspended in 750 μl PBS, mixed with 750 μlsaturated ammonium sulfate solution (final concentration 50% ammoniumsulfate) and centrifuged again. After this, the pellet was suspended in700 μl PBS and dialyzed against the 100 fold volume of PBS. Theresulting IgG fraction can be stored at −20° C. until measurement.

Results

Inhibition of AAB Functionality of Different AABs Against G-ProteinCoupled Receptors by Specific-Aptamers

In the following the neutralization of the activity of antibodies(autoantibodies from patient serum) by the aptamer SEQ. ID No. 1 (alsoknown as thrombin-aptamer SEQ ID No. 1, or ARC 183, described in U.S.Pat. No. 5,543,293) and by the aptamer SEQ. ID No. 2 (also known asthrombin-aptamer SEQ ID No. 2, or ARC 2172 or NU 172, described inWO/2007/025049) was investigated (Table 1).

Doing this the following antibodies (autoantibodies=AAB) wereinvestigated: the adrenergic alpha 1-receptor AAB, the adrenergicbeta1-receptor AAB, beta2-receptor AAB, the muscarinic M₂-receptor-AAB,the endothelin 1 ETA receptor-AAB (ETA-AAB), the angiotensin IIAT1-receptor-AAB, and the PAR-receptor AAB.

The occurrence of such AAB has been described in the followingpathological situations, not excluding other pathological situationswhich might also be carriers of the same or similar AABs: DCM, Chagas'cardiomyopathy, chronic Chagas' disease, Chagas' megacolon, Peripartumcardiomyopathy, Glaucom, pulmonary hypertension hypertension,hypertension, maligne hypertension, diabetis mellitus, Alzheimerdisease, kidney allograft rejection, Raynaud Syndrome. Table 1 showsthat the functional activity of all the tested autoantibodies directedagainst G-protein coupled receptors was neutralized using the aptamerwith SEQ. ID No. 1 or partially neutralized using the aptamer with SEQID No. 2.

TABLE 1 Shows the capacity of the aptamers SEQ. ID No. 1 and SEQ. ID No.2 [100 nM] on the neutralization of the functional activity of AABsdirected against G-protein coupled receptors. The functional activity ofthe AAB was measured via their capacity to change the pulse rate of thecells [Δ beat rate/15 sec]. +Thrombin- +Thrombin- aptamer aptamer Affi-Without (SEQ. ID +scramble (SEQ. ID disease AAB-Typ Loop purifiedaptamer No. 1) aptamer No. 2) DCM Beta1- 1. Patient 1 6.33 −0.17receptor- Loop Patient 2 5.33 0.5 AAB Beta1- 2. Patient 1 5.33 0.17 5.33receptor- Loop 0.33 AAB Glaucom Beta2- 2. Affi Patient 1 7.33 0.33 5.33receptor- Loop Patient 2 8.67 −0.17 AAB Patient 3 6.0 0.67 Patient 47.67 −0.17 Peripartum- Beta1- 2. Affi Patient 1 6.33 0.5 4.0cardiomyopathy receptor- Loop AAB Pulmonary Alpha1- 2. Patient 1 5.5−0.17 2.0 Hypertension receptor Loop Patient 2 5.17 0.33 AAB Endothelin-2. Patient 1 −4.83 −0.17 2.0 receptor- Loop Patient 2 −4.8 0.33 AABHypertension Alpha1- 2. Affi Patient 1 5.5 1.0 3.33 receptor- LoopPatient 2 6.33 −0.83 AAB Malignant. AT1- 2. Patient 1 7.83 0.67Hypertension receptor- Loop AAB Chagas Beta2- 2. Patient 1 6.17 −0.5 2.0Megacolon receptor- Loop AAB Chagas Beta1- 2. Patient 2 3.5 0.5 3.0Cardiomyopathy receptor- Loop AAB M2- 2. Patient 2 −3.67 0.5 3.0receptor- Loop AAB Kidney AT1- 2. Affi Patient 1 6.17 0.17 transplant.receptor- Loop AAB Diabetes Alpha1- Patient 1 3.97 0.0 mellitusreceptor- Patient 2 5.5 0.33 AAB Alzheimer Alpha1-/ 2. Patient 1 8.0 0.0disease beta2- Loop receptor- AAB Beta2- 1. Affi Patient 2 4.67 0.67receptor- Loop AAB Raynaud ETA- Patient 1 −5.17 0.33, 0.0, Syndromereceptor AAB PAR- Patient 1 7.33 0.17 receptor- Patient 2 4.67 −0.33,0.0 AAB

Dose-Response Curves of the Thrombin-Aptamer Mediated AAB-Neutralization

In the following the dose-response curves of the singlethrombin-aptamer-incubations were measured (FIG. 1). Doing this not onlyhuman AABs against G-protein coupled receptors were neutralized, butalso beta1-receptor AABs from the blood of SHR-rats. In fact, human AABsagainst the first or second loop of the beta1-receptor, AABs against theAT1-receptor, and the adrenergic alpha1-receptor were neutralized. Thebeta1-receptor AAB in rats is a spontaneously formed AAB.

It became quite obvious that the different dose-response curves showedslightly differences. While the neutralization effect was most efficientfor the human alpha 1-receptor AAB, it showed the smallest efficiencyfor the rat-beta1-receptor AAB. Taken the AAB concentration might bearound 0.1% of the IgG fraction (personal information Dr. Wallukat) thanthe different aptamer concentrations would face a final concentration ofabout 1.4 nM AAB (normal IgG concentration about 10 g/1=68.5 μM, AABdilution in the cell cultivation medium 1:50). This way it becomes verylogical that the neutralization effect starts at some AAB such as thealpha 1-receptor AAB at an aptamer concentration of about 1 nM, while ingeneral 100 nM aptamer are necessary for a complete inhibition of theAAB functionality.

Aptamer—AAB Affinity in the Presence of Competing Human IgG-3

Former experiments testing the affinity of the thrombin-aptamer SEQ IDNo. 1 towards the human IgG-subtypes had shown that the thrombin-aptamerSEQ ID No. 1 had a higher affinity towards IgG-3 followed by IgG-4,IgG-2, and IgG1. While the differences between the last three subtypeswere only marginal, the affinity towards the subtype IgG-3 was striking(data not shown).

Now, in order to test, if the affinity of the thrombin-aptamer SEQ IDNo. 1 towards the AAB is higher compared to its common affinity towardsthe IgG-3 subclass, the following competition experiment was carriedout: while measuring the dose-response curve of the thrombin-aptamer SEQID No. 1 against a human patient serum AAB (beta1-receptor AAB, secondloop, dilution 1:50), in one experimental set 73 nM human IgG-3 wasadded allowing to compete with the beta1-receptor AAB about thethrombin-aptamer-binding (FIG. 2). Especially at the low concentrationof the thrombin-aptamer SEQ ID No. 1 (1, 5, and 10 nM thrombin-aptamer)at which the IgG-3 concentration is clearly in the molar excess to thethrombin-aptamer, no differences were observed betweenAAB-neutralization effects, with and without the presence of IgG-3.

Demonstration of the Binding Between the Thrombin-Aptamer and theAutoantibody Using a Model Autoantibody: RabbitAnti-Human-Endothelinreceptor Antibody Against the Second ExtracellularLoop of the Receptor (Acris Antibodies, SP 41222P)

1. ETA-AB Functionality Testing in the Bioassay and Neutralization bythe Thrombin-Aptamer SEQ ID No. 1

The rabbit endothelinreceptor antibody (ETA-AB) was generated againstthe second extracellular loop of the human endothelin receptor. Theantibody showed functional activity in the bioassay. The ETA-AB caused areduction of the beating frequency of spontaneously beating neonatalerat cardiomyocytes (FIG. 3). The addition of 100 nM thrombin-aptamercaused the complete neutralization of this ETA-AB caused change in thebeating frequency (FIG. 3). The addition of the thrombin-aptamer (SEQ.No. 1) alone onto the neonatale rat cardiomyocytes did nor cause anyvisual effect onto the cells nor did it influence on the basal beatingfrequency (data not shown).

2. Testing the Direct Binding Between the Thrombin-Aptamer SEQ ID No. 1and the ETA-AB

Testing the direct binding between the thrombin-aptamer and the ETA-ABin the ELISA-experiment two different experimental set-ups were tested.Firstly the ETA-AB was immobilised onto the ELISA plate and its abilityto bind the thrombin-aptamer SEQ ID No. 1 was tested (FIGS. 4A-4D). In asecond set of experiments the thrombin-aptamer SEQ ID No. 1 wasimmobilised and the ETA-AB was offered for binding (FIGS. 5A, 5B).

For the first set of experiments two different protein-concentrationsfor the immobilization onto the ELISA plate were chosen: 25 nM (FIG. 4A)and 250 nM (FIG. 4B). For control served rabbit IgG, human-IgGsubclasses (IgG-1,2,3 and 4) at the same concentrations, and theuncoated plastic plate. A further control was the scrambledthrombin-aptamer sequence which was offered for binding (FIGS. 4C, 4D).

A second set of experiments tested the binding between the ETA-AB andthe thrombin-aptamer SEQ. ID No. 1 in a vice versa experiment. Now thebiotinylated thrombin-aptamer was immobilized onto the ELISA plates viapreimmobilization of neutravidine. Afterwards the ETA-AB was offered forbinding (FIGS. 5A, 5B).

Comparing the extinction between FIGS. 5B and 5A shows that a coating of0.1 μM thrombin-aptamer (SEQ. ID No. 1) reached already the saturationin the given experiment.

A further control was the binding of the ETA-AB onto the immobilizedscrambled thrombin-aptamer (FIG. 6).

The affinity of the ETA-AB towards the scrambled thrombin-aptamer wasabout 50% of the affinity reached if the thrombin-aptamer (SEQ. IDNo. 1) was offered for binding.

Thombinaptamer-Column for the Removal of AAB from Serum Rabbit ETA-ABSpiked Human Serum

In the following a thrombin-aptamer column was made which served for theremoval of AAB from serum. For control served a column which carried thescrambled thrombin-aptamer.

The aptamers (thrombin-aptamer SEQ. ID No. 1) and scrambled controlsequence were bound onto column material (NHS-activated Sepharose, GEhealthcare) at a concentration of 0.1 μmol.

In a first set of experiments serum was spiked with the rabbit ETA-AB(50 nM) in order to obtain the chance to not only measure the ETA-ABactivity via the bioassay (FIG. 7) but also via an ELISA (FIGS. 8A, 8B).The spiked sera were given over the column and the control column. Theflow through was taken and stored. The bound ETA-ABs were eluted using a3 M KSCN solution. Before measuring the AAB activity in the eluates, thesamples were dialysed against physiologic buffer for 3 days at 4° C.

In the bioassay the eluates, which were taken in two fractions, weremeasured (FIG. 7). While the thrombin-aptamer column showed the ETA-ABactivity after elution, the control column did not. The main ETA-ABfraction was in the second eluate fraction which was owed the usedvolumes. The volume of the column was 500 μl while for all handlingsteps 250 μl were chosen.

For the detection of the ETA-AB in the flow through or the eluate of thecolumns in the ELISA, the ETA-AB standard curve of the ELISA had also toundergo the dialyzation procedure, comparable to the eluate-samplematerial (FIG. 8A). Using this standard material, the samples weretested for the ETA-AB presence (FIG. 8B).

It is shown that only the specific thrombin-aptamer-column was able tobind the ETA-AB from the spiked control serum, while the scrambledcontrol aptamer did not bind the ETA-AB. Here the ETA-AB was found inthe flow through, while with the specific column the eluate fractionscontained the ETA-AB.

Just to exclude that bound human IgG from the control serum might viacross reaction with the secondary antibody mimic ETA-AB presence, serum(40% and 100%) was also applied onto the ELISA plate. The maximalpossible amount of cross reaction was measured, which was smaller thanthe specific anti-rabbit-AB detection. Moreover, it was shown inindependent experiments that the eluates contained less than 1/10 humanIgG compared to the flow through samples (data not shown), excluding anenormous influence of secondary antibody cross reactivity.

2. Human AAB Containing Serum

In a second series of experiments the thrombin-aptamer column was usedfor the removal of serum AAB. For control served the scrambledthrombin-aptamer column.

For this purpose 250 μl ETA-AAB and alpha1-receptor AAB containing serumwas given over the columns. The flow throughs and the eluates werecaptured and estimated for AAB activity using the bioassay (Tab. 2).Elution was done with 3 M KSCN. The samples were dialyzed against aphysiologic buffer for 3 days before the AAB-activity measurement wascarried out.

TABLE 2 Measurement of the ETA-AAB and the alpha1-receptor AAB activityfrom human serum in the single fractions of the column experimentControl column Decrease/increase Thrombin- (scrambled in bead numberaptamer- tThrombin- [beads/15 sec] column aptamer) Flow through Alpha1-receptor- −0.17 6.17 AAB Endothelin-receptor- −0.17 −4.17 AAB 1steluate Alpha 1-receptor- 2.0 0.17 AAB Endothelin-receptor- −1.83 0.17AAB 2nd eluate Alpha 1-receptor- 4.67 −0.17 AAB Endothelin-receptor-−2.5 −0.17 AAB 3rd eluate Alpha 1-receptor- 1.0 n.d. AABEndothelin-receptor- −0.5 n.d. AAB n.d. = not determined

Possible Kit for Purification of Serum from AAB Via Aptamer-MagneticBeads

A kit for either purification of serum from AAB or for theAAB-enrichment via thrombin-aptamer-magnetic beads.

TABLE 3 Binding of serum-AAB (human and rat) onto immobilizedthrombin-aptamer (SEQ. ID No. 1; Streptavidin magnetic beads andbiotinylated thrombin-aptamer) Thrombinapta Thrombinapta Delta Beads/(SEQ. ID No. 1) Scram-Thrombin (SEQ. ID No. 1) 15 sec human ß1-AAB Humanß1-AAB SHR-rat Particle 0 5.5 0 supernatant 1. Washing 0 n.d. 0 solution1st eluate 5.67 1.0 (1st + 2nd 4.67 (beta1- (beta1receptor- eluatecombined) receptor-AAB) AAB) −1.83 (M2-receptor- AAB) 2nd eluate 2.5(beta1- 2.5 (beta1-receptor- receptor- AAB) AAB) −2.83 (M2-receptor-AAB)

Introduction of Exonuclease-Protection

In the following the influence of the introduction of a 3′-dT cap ontothe functional activity of the aptamer SEQ. ID No. 1 was tested. A 3′-dTcap is thought to protect the nucleotide sequence from exonucleaseactivity and increases its stability in biological fluids. Testing theeffect of the 3′-dT cap onto the aptamer functionality thebeta1-receptor AAB (2. Loop) from a DCM patient and the beta2-receptorAAB from an Alzheimer patient were used (Tab 4). The functional activityof both AABs was neutralized when incubated with 100 nM of the 3′-dT capmodified aptamer. The 3′-cap-modification did not influence thefunctional activity of the aptamer SEQ. ID No. 1.

The CAP-protected aptamer alone did not influence the basal beating rateof the neonatale cardiomyocytes.

TABLE 4 Bioassay-measurement of the serum AAB chronotropic activity[delta beads/15 sec] of AABs treated with 3′-dT-cap modifiedthrombin-aptamer SEQ. ID No. 1 or not (without aptamer). Without+aptamer-3′-dT disease AAB-Typ aptamer cap DCM Beta1-receptor-AAB 6.00.0 Alzheimer Beta2-receptor-AAB 5.17 0.0

FITC-Labelled Thrombin-Aptamer for the Detection of AABs

Use of Directly Fluorescence-Marker Labelled Thrombin-Aptamer for theDetection of Serum AABs

The aim of these experiments is the generation of a directly labelledaptamer which targets autoantibodies against G-protein coupled receptorswhich will, at the end, be exploited for the detection of such AABs.

For this purpose the thrombin-aptamer was labeled at the 5′-end withFITC.

In first experiments it was tested, if the FITC-labelledthrombin-aptamer showed the full functionality/activity to neutralizeautoantibodies compared to its unlabelled version. This was tested usingthe bioassay.

FIG. 9 shows the capacity of 100 nM of the 5′-FITC-thrombin-aptamer toneutralize beta1-receptor AABs. The FITC-label reduced the activity ofthe thrombin-aptamer, but 50% of the beta1-receptor AAB-activity wasstill neutralized at this chosen concentration of 100 nM. A partialinhibition of AAB activity was observed at this aptamer concentration.

Since the FITC-labelled thromnin-aptamer showed—if compared with theunlabeled aptamer at the same concentration—a partial inhibition of theAAB activity, the molecule was taken for testing, if it would be apossible strategy to use this labelled thrombin-aptamer for a sandwichassay. For this reason the unlabelled biotinylated thrombin-aptamer wasimmobilised on the ELISA-plate via Neutravidin and served for catchingthe AABs while the FITC-labelled anti.-thrombin-aptamer was supposed toserve at the end for the detection of the adsorbed AAB-material.

The samples were removed from the wells, the duplicates were unified(final volume 200 μl) and diluted with 300 μl PBS and the relativefluorescence was measured using the spectrofluorophotometer RF-5001PC(Shimadzu, Japan) using the excitation and emission wavelength of 494 nmand 519 nm, respectively.

As to be seen from FIG. 10 it was possible to detect a patient sample(IgG-fraction containing ETA-AABs) compared to control IgG-sample. Usingthe scrambled thrombin-aptamer as a further control did also not showany fluorescence.

Influence of the dT-Cap on the Functionality of the Thrombin-Aptamer

In next experiments it was tested, if the introduction of a protectinggroup such as the dT-cap for the protection from exonuclease activitywould be possible, without influencing on the thrombin-aptamer activityneutralizing autoantibodies against G-protein coupled receptors. Theintroduction of a dT-cap was possible without influencing thethrombin-aptamer functionality to neutralize AABs against G-proteincoupled receptors (FIG. 11).

Truncation of the Thrombin-Aptamer Resulting in 12Mer Sequence

In a next set of experiments it was tested if a truncation of thethrombin-aptamer would result in products which are still able toneutralize autoantibodies against G-protein coupled receptors.

The original 15-mer sequence of the thrombin-aptamer (ARC183) was,therefore, truncated into a 12-mer sequence with SEQ D No. 3, calledThrom-K1. The truncated sequence was tested for its ability toneutralize AABs in the bioassay (demonstrated for beta1-receptor AABs,FIG. 12).

The 12-mer sequence showed full activity.

1. A method of treatment or diagnosis of autoimmune diseases, wherein anindividual suffering from an autoimmune disease is administered aneffective amount of an aptamer comprising a nucleic acid sequence of SEQID No. 1, SEQ ID No. 2, SEQ ID No. 3 and/or a nucleic acid sequencebeing at least 80% identical to one of SEQ ID No. 1, 2 and 3 whereinautoantibodies specific for a G-protein coupled receptor are present inthe serum of said individual suffering from said autoimmune disease. 2.The method of claim 1, wherein the autoimmune disease is selected fromthe group consisting of: cardiomyopathy, dilated cardiomyopathy (DCM),peripartum cardiomyopathy (PPCM), idiopathic cardiomyopathy, Chagas'cardiomyopathy, Chagas' megacolon, Chagas' megaesophagus, Chagas'neuropathy, benign prostatic hyperplasia, scleroderma, psoriasis,Raynaud syndrome, pre-eclampsia, kidney allograft rejection,myocarditis, glaucoma, Diabetes mellitus, hypertension, pulmonaryhypertension, malignant hypertension and Alzheimer's disease.
 3. Themethod of claim 1, wherein an aptamer comprising the nucleic acidsequence of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 and/or a nucleicacid sequence being at least 80% identical to one of SEQ ID No. 1, 2 and3 is used as a selective ingredient during apheresis.
 4. The method ofclaim 3, wherein the aptamer is used during therapeutic apheresis ofblood or constituents thereof, of a patient suffering from an autoimmunedisease.
 5. The method of claim 4, wherein the autoimmune disease isselected from the group consisting of: cardiomyopathy, dilatedcardiomyopathy (DCM), peripartum cardiomyopathy (PPCM), idiopathiccardiomyopathy, Chagas' cardiomyopathy, Chagas' megacolon, Chagas'megaesophagus, Chagas' neuropathy, benign prostatic hyperplasia,scleroderma, psoriasis, Raynaud syndrome, pre-eclampsia, kidneyallograft rejection, myocarditis, glaucoma, hypertension, pulmonaryhypertension, malignant hypertension, and Alzheimer's disease.
 6. Themethod of claim 1, wherein the aptamer is used in treatment of a human.7. The method of claim 1, wherein the aptamer is used in diagnosis of ahuman.
 8. The method of claim 1, wherein the aptamer is used intreatment and diagnosis of a human.
 9. The method of claim 1, whereinthe aptamer is a DNA aptamer.
 10. The method of claim 1, wherein theaptamer consists of the nucleic acid sequence of SEQ ID No. 1, SEQ IDNo. 2, SEQ ID No. 3 or a nucleic acid sequence being at least 80%identical to one of SEQ ID No. 1, 2 and
 3. 11. The method of claim 1,wherein the aptamer is administered as a pharmaceutical compositioncomprising the aptamer of claim 1 and at least one pharmaceuticallyacceptable excipient.
 12. The method of claim 1, wherein the aptamer isprovided as a kit comprising the aptamer of claim 1 and a container. 13.The method of claim 1, wherein the autoimmune disease is associated withthe presence of autoantibodies specific for a G-protein coupledreceptor.